Administration of supraphysiological concentration of oxygen (hyperoxia) is a common clinical practice in the management of premature babies and adults with respiratory failures. However, prolonged exposure to hyperoxia is toxic to lung tissue, causing injury and mortality. The mechanisms of hyperoxic lung injury are not well defined, however, elevated levels of reactive oxygen species (ROS) and reactive nitrogen species with decreased alveolar glutathione may account for the pulmonary toxicity, endothelial damage, and vascular leakiness. Damage to the endothelium results likely from ROS generated by neutrophil-dependent and neutrophil-independent enzymatic pathways of NAD(P)H oxidase, xanthine oxidase, cycloxygenase/lipoxygenase, and mitochondrial electron transport. We have identified activation of endothelial NAD(P)H oxidase and enhanced production of ROS in hyperoxia, however, there remains several unanswered questions regarding the role and regulation of endothelial NAD(P)H oxidase in vascular injury and leakiness. It is hypothesized that acute lung injury and barrier dysfunction due to hyperoxia are mediated by ROS generated by the activation of endothelial NAD(P)H oxidase system. To test this hypothesis, we will investigate the effect of hyperoxia in NAD(P)H oxidase activation ROS generation, cytoskeletal changes and barrier dysfunction in human endothelial cells and in animal models. SA1: To characterize hyperoxia-mediated O2/ROS generation by endothelial NAD (P)H oxidase. SA2: To investigate the role of protein kinases in hyperoxia-induced NAD(P)H oxidase activation. SA3: To characterize the role of phospholipase D/phosphatidic acid in hyperoxia-mediated NAD(P)H oxidase activation. SA4: To determine the role of hyperoxia-mediated NAD(P)H oxidase in cytoskeletal re-arrangement and endothelial barrier dysfunction/vascular leakiness. Further understanding the role and regulation of endothelial NAD(P)H oxidase activation in hyperoxia will allow development of targeted therapies to attenuate ROS-induced lung injury and reverse endothelial dysfunction.